Overall, the projects in our laboratory focus on the inflammatory, angiogenic, immunological and algesic consequences of chemokine receptor interactions with chemokines and with chemokine mimics such as defensins, other antimicrobial peptides autoantigens and some tumor antigens. We have previously shown that defensins are chemoattractants for antigen presenting immature dendritic cells (iDC). Beta defensins interact with CCR6, while alpha defensins use an as yet unidentified GiPCR. This has led us to identify other ligands interacting with iDC in an effort to identify even more potent immune adjuvants. In collaboration with Drs. Arya Biragyn and Larry Kwak, we have shown that tumor antigens linked to defensins or chemokines become more immunogenic. Using fusion products of murine beta defensin 2 (mBD2) and immunoglobulin tumor antigens as vaccines protects mice against tumors, because mBD2 also interacts with TLR4 to activate iDC. Pertussis toxin (PTX) and the B chain of PTX, which are well-known immunoadjuvants, induce iDC to mature by interacting with TLR4. Eosinophil derived neurotoxin (EDN), an RNA'se with anti-HIV activity, is also chemotactic for iDC, and induces maturation of DC with the expression of costimulatory molecules, MHC class II markers and production of inflammatory cytokines. High Mobility Group Box 1 (HMGB1), a DNA binding protein induces inflammatory cytokine production and matures iDC according to Dr. Kevin Tracey and coworkers and is chemotactic for iDC in our hands. Cathelicidin (LL37) is chemotactic for phagocytic cells and T cells, but not iDC. LL37 also has adjuvant activity and we plan to test the tumor vaccine efficacy of several of the "endogenous multifunctional immune alarmins" by linking them to tumor antigens. These findings enabled us to appreciate Dr. Zack Howard's observations that autoantigens frequently are chemotactic for GiPCR expressing mononuclear cells which may account for their immunogenicity. Histidyl tRNA Synthetase (HRS) and AsnRS, autoantigens involved in myositis, are chemotactic for iDC and other cells expressing CCR5 and CCR3 respectively. Amino acid tRNA synthetases that are not autoantigens are not chemotactic. Of the antigens that induce experimental autoimmune uveitis (EAU): IRBP uses CXCR5 and CXCR3; S-antigen uses CXCR3. Some tumor antigens are also chemotactic for iDC e.g. GP100 and MUC-1, but not NY-ESO-1. In contrast with EDN, gp100 does not induce normal iDC to mature or to produce proinflammatory cytokines. We hypothesize that secreted/released tumor antigens are "normally" tolerogenic and plan to convert them to immunogenic signals. We propose to identify the extracellular signals (e.g. cell contact dependent and or soluble mediators) that induce tolerance. Our laboratory has previously shown that a number of chemokines are pro-angiogenic. Inhibitors (anti-MCP-1, anti SDF1 alpha and anti-IL8 together with anti-EGF) interfere with tumor vascularization and growth. SDF-1 - CXCR4 interactions have the most potent angiogenic effects by inducing VEGF production and conversely VEGF and betaFGF by inducing PGE2,via the COX-2 more than the COX-1 pathway, upregulate CXCR4 expression. Inhibitors of COX-2 >COX-1 production of PGE2 inhibit CXCR4 expression on endothelial cells and conversely, anti-CXCR4 blocks most of the angiogenic effects of PGE2 and about 50% of the effect of VEGF/bFGF. Anti CXCR4 is reported to reduce breast tumor metastasis in mice. Dr. Zack Howard has obtained some preliminary evidence that local administration of anti SDF-1 into tumors has antiangiogenic effects. Furthermore, we are collaborating with Dr. Chris Mijheda in evaluating the antitumor effects of a more potent peptide inhibitor of CXCR4. Dr. Zack Howard and Xin Chen are identifying other inhibitors of chemotactic activities present in natural products, synthetic compounds and Chinese Traditional Medicines to identify anti-inflammatory agents with anti-angiogenic and potential anti-tumor effects. We previously have shown that a synthetic compound suradista NSC 65016 inhibited the chemotactic response of CXCR4 expressing monocytes to its ligand, SDF1/CXCL12. We determined that this compund also interfered with SDF1 induced endothelial cell (EC) chemotaxis, new vessel sprouting, capillary-tubal formation by EC and in vivo neovascularization. Thus, NSC651016 is also antiangiogenic and may have antitumor activity. Two traditional Chinese medicinals (TCM) with known antiinflammatory activities, Qiukailian (QKL) and Shuanghuanglian (SHHL) had the capacity to suppress NFKB regulated gene transcription including reduced production of cytokines and chemokines. These TCM were not cytotoxic, but also inhibited the migratory response of leukocytes to chemokines. The responsible molecular components need to be purified and identified. Pain as a host defense is induced in the course of inflammation. Chemokines augment the in vivo perception of pain in the CNS by "cross-desensitizing" opioid receptors, via non-classical PKC dependent phosphorylation of opioid receptors. We therefore hypothesized that chemokines may have the opposite effect of "sensitizing" peripheral pain receptors. This led Dr. Ning Zhang to show that chemokines like MIP1 alpha acting on CCR1, expressed on small neurons in the dorsal root gaglion (DRG), sensitize and enhance the response of co-expressed VR1 pain receptors on DRG neurons. In fact, prior exposure to MIP1 alpha enhances the vanilloid receptor (VR1) calcium flux in response to capsaicin by three to five -fold. Thus inflammation enhances pain via receptor cross-talk. We plan to determine which other chemokines and cytokines have this sensitizing capacity and whether this mechanism also affects other pa